3-3-di-substituted-oxindoles as inhibitors of translation initiation

ABSTRACT

Compositions and methods for inhibiting translation using 3-(5-tert-Butyl-2-Hydroxy-phenyl) -3-phenyl-1,3-dihydro-indol-2-one and/or its derivatives are provided. Compositions, methods and kits for treating (1) cellular proliferative disorders, (2) non-proliferative, degenerative disorders, (3) viral infections, and/or (4) disorders associated with viral infections, using 3-(5-tert-butyl-2-hydroxy-phenyl)-3-phenyl-1,3-dihydro-indol-2-one and/or its derivatives are described.

RELATED U.S. APPLICATION(S)

This application is a continuation of U.S. application Ser. No.11/463,421 filed on Aug. 9, 2006 which claims the benefit of PCTapplication no. PCT/US2005/004373, designating the United States andfiled Feb. 11, 2005; which claims the benefit of the filing date of U.S.provisional application No. 60/544,384, filed Feb. 13, 2004; all ofwhich are hereby incorporated herein by reference in their entirety.

STATEMENT OF GOVERNMENT INTERESTS

This invention was made with Government support under Grant Number 5 U19CA87427 awarded by the National Institutes of Health. The Government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to novel compounds which inhibittranslation initiation, pharmaceutical compositions of the novelcompounds, and methods of treating medical disorders.

BACKGROUND OF THE INVENTION

Translation, the mRNA-directed synthesis of proteins, occurs in threedistinct steps: initiation, elongation and termination. Translationinitiation is a complex process in which the two ribosomal subunits andmethionyl tRNA (met-tRNA) assemble on a properly aligned mRNA tocommence chain elongation at the AUG initiation codon. The establishedscanning mechanism for initiation involves the formation of a ternarycomplex among eukaryotic initiation factor 2 (eIF2), GTP and met-tRNA.The ternary complex recruits the 40S ribosomal subunit to form the 43Spre-initiation complex. This complex recruits mRNA in cooperation withother initiation factors such as eukaryotic initiation factor 4E(eIF4E), which recognizes the 7-methyl-guanidine cap (m-⁷GTP cap) in anmRNA molecule and forms the 48S pre-initiation complex. Cap recognitionfacilitates the 43S complex entry at the 5′ end of a capped mRNA.Subsequently, this complex migrates linearly until it reaches the firstAUG codon, where a 60S ribosomal subunit joins the complex, and thefirst peptide bond is formed (Pain (1996) Eur. J. Biochem.,236:747-771).

Several features of the mRNA structure influence the efficiency of itstranslation. These include the m-⁷GTP cap, the primary sequencesurrounding the AUG codon and the length and secondary structure of the5′ untranslated region (5′ UTR). Indeed, a moderately long, unstructured5′ UTR with a low G and C base content seems to be optimal to ensurehigh translational efficiency. Surprisingly, sequence analysis of alarge number of vertebrate cDNAs has shown that although mosttranscripts have features that ensure translational fidelity, many donot appear to be designed for efficient translation (Kozak (1991) J.Cell. Biol., 115:887-903). Many vertebrate mRNAs contain 5′ UTRs thatare hundreds of nucleotides long with a remarkably high GC content,indicating that they are highly structured because G and C bases tend toform highly stable bonds. Because highly structured and stable 5′ UTRsare the major barrier to translation, mRNAs with stable secondarystructure in their 5′ UTR are translated inefficiently and theirtranslation is highly dependent on the activity of translationinitiation factors.

mRNAs with complex, highly structured 5′ UTRs include adisproportionately high number of proto-oncogenes such as the G1cyclins, transcription and growth factors, cytokines and other criticalregulatory proteins. In contrast, mRNAs that encode globins, albumins,histones and other housekeeping proteins rarely have highly structured,GC-rich 5′ UTRs (Kozak (1994) Biochimie, 76; 815-21; Kozak (1999) Gene,234:187-208). The fact that genes encoding for regulatory but not forhousekeeping proteins frequently produce transcripts with highlystructured 5′ UTRs indicates that extensive control of the expression ofregulatory genes occurs at the level of translation. In other words, lowefficiency of translation is a control mechanism which modulates theyield of proteins such as cyclins, mos, c-myc, VEGF, TNF, among others,that could be harmful if overproduced.

SUMMARY

Translation initiation is a critical step in the regulation of cellgrowth because the expression of most oncogenes and cell growthregulatory proteins is translationally regulated. One approach toinhibiting translation initiation has recently been identified usingsmall molecule known as translation initiation inhibitors. Withoutintending to be bound by theory, FIG. 1 sets forth a summary of theanti-cancer mechanism of action of translation initiation inhibitorssuch as clotrimazole (CLT) and the diaryloxindole (DAO) compounds of thepresent invention. CLT inhibits translation initiation by sustaineddepletion of intracellular Ca²⁺ stores. Depletion of intracellular Ca²⁺stores activates “interferon-inducible” “double-stranded RNA activated”protein kinase (PKR) which phosphorylates and thereby inhibits the αsubunit of eIF2. Since the activity of eIF2 is required for translationinitiation, its inhibition by compounds such as CLT reduces the overallrate of protein synthesis. Because most cell regulatory proteins areencoded for by mRNAs containing highly structured 5′ UTRs, they arepoorly translated and their translation depends heavily on translationinitiation factors such as eIF2 and eIF4. Therefore, inhibition oftranslation initiation preferentially affects the synthesis andexpression of growth regulatory proteins such as G1 cyclins. Sequentialsynthesis and expression of G1 cyclins (D1, E and A) is necessary todrive the cell cycle beyond the restriction point in late G1. Thus, thedecreased synthesis and expression of G1 cyclins resulting fromCLT-induced inhibition of translation initiation causes cell cyclearrest in G1 and inhibits cancer cell and tumor growth (Aktas et al.(1998) Proc. Natl. Acad. Sci. USA, 95:8280-8285, incorporated herein byreference in its entirety for all purposes).

Like CLT, the n-3 polyunsaturated fatty acid eicosapentaenoic acid (EPA)depletes internal calcium stores, and exhibits anti-carcinogenicactivity. Unlike CLT, however, EPA is a ligand of peroxisomeproliferator-activated receptor gamma (PPARγ), a fatty acid-activatedtranscription factor. Although EPA and other ligands of PPARγ, such astroglitazone and ciglitazone, inhibit cell proliferation, they do so ina PPARγ-independent manner (Palakurthi et al. (2000) Cancer Research,60:2919; and Palakurthi et al. (2001) Cancer Research, 61:6213,incorporated herein by reference in their entirety for all purposes).

Embodiments of the present invention are directed to compounds thatinhibit translation initiation, and the use of such compounds fortreating (1) proliferative disorders, (2) non-proliferative,degenerative disorders, (3) viral infections, and/or (4) disordersassociated with viral infections are disclosed. Certain examples aredirected to the use of a combination of the compounds described hereinfor treating (1) proliferative disorders, (2) non-proliferative,degenerative disorders, (3) viral infections, and/or (4) disordersassociated with viral infections.

In at least certain examples, DAO compounds are provided. The terms“diaryloxindole” AND “DAO” are intended to include substituteddiphenyloxindole compounds such as, for example,3-(5-tert-butyl-2-hydroxy-phenyl)-3-phenyl-1,3-dihydro-indol-2-one-typecompounds. In one example, diaryloxindole compounds of the presentinvention are set forth in Formula I. In certain examples, thediaryloxindole compounds of the present invention deplete intracellularcalcium stores. In another example, diaryloxindole compounds areeffective to inhibit translation initiation. In one aspect, thediaryloxindole compounds of the present invention include compoundscomprising Formula I to Formula XIV, and/or the active and partiallyactive compounds set forth in Tables 1-14 and FIGS. 4A-4B.

In accordance with a method aspect, a method of treating a proliferativedisorder by providing and/or administering a diaryloxindole compound toa mammal, e.g., a human or a non-human (e.g., a non-human primate), isprovided. In one example, the proliferative disorder is cancer. Inaccordance with other examples, a method of treating a viral infectionby providing and/or administering a diaryloxindole compound to a mammal,e.g. a human or a non-human mammal, is provided.

In accordance with an additional aspect, kits are provided for thetreatment of (1) proliferative disorders, (2) non-proliferative,degenerative disorders, (3) viral infections, and/or (4) disordersassociated with viral infections. In one aspect, the kits comprise adiaryloxindole compound, a pharmaceutically acceptable carrier, andoptionally, instructions for use. The pharmaceutical composition can beadministered to a human subject or a non-human subject depending on thedisorder to be treated.

It will be recognized by the person of ordinary skill in the art thatthe compounds, compositions, methods and kits disclosed herein providesignificant advantages over prior technology. Compounds, compositions,methods and kits can be designed or selected to relieve and/or alleviatesymptoms in a patient suffering from one or more disorders. These andother aspects and examples are described below.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee. The foregoing and other features and advantages ofthe present invention will be more fully understood from the followingdetailed description of illustrative embodiments taken in conjunctionwith the accompanying drawings.

FIG. 1 depicts a schematic of the anti-cancer mechanism of action fortranslation initiation inhibitors such as clotrimazole (CLT) anddiaryloxindole (DAO).

FIGS. 2A-2B depict compound 1181 activity. (A) depicts calcium (Ca²⁺)release in a dose dependent manner as compared to CLT from intracellularstores measured using dye-loaded cells. (B) depicts depletion ofendoplasmic reticulum (ER) Ca²⁺ in stable cells lines carryingER-targeted Ca²⁺-sensitive chameleon proteins exposed to compound 1181,in accordance with certain examples. These proteins emit light and theCa²⁺ content is measured by fluorescent resonance energy transfer(FRET).

FIGS. 3A-3C depict compound 1181 activity. (A) depicts the drug inducedphosphorylation of eIF2α protein in control (lane 1), positive control(lane 2) and 1181 (lane 3) measured using Western blot analysis. (B)shows the in vitro anti-cancer effect of 1181 in human lung cancer cells(A549) measured using sulfur rhodamine B (SRB) assay. (C) graphicallydepicts depletion of ternary complex in transgenic KLN cells exposed tocompound 1181.

FIGS. 4A-4B depict a general schematic for the synthesis of peptidesthat contain the diaryloxindole moiety.

FIG. 5 graphically depicts a decrease in squamous cell carcinoma (KLN)tumor mass from DBA/2J mice after six weeks of treatment by oraladministration with compound 1181 at 320 mg/kg/day.

It will be recognized that the results and examples in the figures areonly illustrative and other examples and illustrations will be readilyrecognized by the person of ordinary skill in the art, given the benefitof this disclosure.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

In accordance with certain examples, diaryloxindole compounds thatinhibit translation (e.g., translation initiation) are provided. Suchcompounds are useful for the treatment of (1) proliferative disorders,(2) non-proliferative, degenerative disorders, (3) viral infections,and/or (4) disorders associated with viral infections.

Certain examples are described below with reference to various chemicalformulae. The chemical formulae referred to herein can exhibit thephenomena of tautomerism, conformational isomerism, stereo isomerism orgeometric isomerism. As the formulae drawings within this specificationcan represent only one of the possible tautomeric, conformationalisomeric, enantiomeric or geometric isomeric forms, it should beunderstood that the invention encompasses any tautomeric, conformationalisomeric, enantiomeric or geometric isomeric forms which exhibitbiological or pharmacological activity as described herein.

The compounds and compositions provided below are effective to inhibittranslation (e.g., translation initiation) at least to the extentnecessary for effective treatment of one or more disorders describedherein. While in certain examples translation may be substantiallyinhibited such that little or no activity results, in other examples theinhibition is at least sufficient to relieve and or alleviate thesymptoms from a selected disorder to be treated.

In accordance with certain embodiments, compounds of the invention arerepresented by the generic formula set forth below.

In accordance with certain aspects, R₁ is selected from the groupconsisting of (halo)alky, (un)substituted (alkyl)aryl, halogen, CN,COOH, alkenyl, alkynyl, alkoxy, cycloalkoxy, H, 2-OH, 2-Me, 2-OMe,2-CH₂CO₂H, 2-NHFmoc, 2-NHSO₂CH₂Ph (wherein Ph is phenyl), 2-NHSO₂CH₃,2-NHSO₂(4-tBu)Ph, 2-NHSO₂(4-NHAc)Ph, 2-NHSO₂(3-CF₃)Ph, 2-NHSO₂(4-NO₂)Ph,2-NHSO₂(4-OMe)Ph, 2-NHSO₂(4-Br)Ph, 2-NHSO₂(4-I)Ph, 2-NHSO₂(4-Ph)Ph,2-NHSO₂(4-OPh)Ph, 2-NHAc, 2-NHCO(4-tBu)Ph, 3-O(CH₂)₄NMe₂, 3-O(CH₂)₄N₃,3-t-Bu, 3-OMe, 3-Cl, 3-F, 3-Me, 3-t-Bu, 3′-NHSO₂(4-tBu)Ph, 4-Cl, 4-F,4-Br, 4-OH, 4-Et, 4-O(CH₂)₃CHCH₂, 4-n-propyl, 4-i-Bu, 4-n-Bu,4-O(CH₂)₂OH, 4-OMe, 4-t-Bu, 4-OPh, 4-Ph, 4-NMe₂, 4-ethyl, 5-Br, 5-Cl,5-I, 5-sulfonic acid, 5-nitro, 5-ethyl, 5-fluoro, 5-OCF₃, 5-NO₂, 5-OMe,5-NH₂, 5-Et, 5-F, 5-N₃, 5-NHSO₂-1-napthyl, 6-ethyl, 6-Br, H, 6-Et, 6-Cl,7-ethyl, 7-COOH, 7-COOMe, 7-Br, 7-CF₃, 7-OMe, 7-1 and 7-Et; R₂ isselected from the group consisting of (halo)alky, (un)substituted(alkyl)aryl, halogen, CN, COOH, alkenyl, alkynyl, alkoxy, cycloalkoxy,H, 2-Me and 2-OMe, 2-OH, 3-Cl, 3-F, 3-Me, 4-F, 4-O(CH₂)₃CHCH₂, 4-OH,4-t-Bu, 4-O(CH₂)₃CHCH₂, 7-formamide-N-[2-(4-Amino-phenyl)-ethyl],7-COOH, 7-CH₂OH and 7-CH₂—N-imidazole; R₃ is selected from the groupconsisting of (un)substituted aryl, (un)substituted heterocyclic,(un)substituted

heteroaromatic, Ar—NHSO₂Ar, Ar—NHCOAr, H,

2-thiophene, —(CH₂)₁₁CH₃, —CH₂Ph, —CCH, -cyclohexyl and —C₆F₅, wherein Xis selected from the group consisting of H, 2-OH, 2-OMe, 2-Me, 2-OMe,2-OH, 2-CH₂CO₂H, 3-OMe, 3-Cl, 3-F, 3-Me, 3-O(CH₂)₄NMe₂, 3-O(CH₂)₄N₃,3-t-Bu, 4-F, 4-O(CH₂)₃CHCH₂, 4-OH, 4-n-propyl, 4-i-Bu, 4-n-Bu,4-O(CH₂)₂OH, 4-OMe, 4-t-Bu, 4-OMe, 4-OPh, 4-Ph, and 4-NMe₂; R₄ isselected from the group consisting of (un)substituted aryl,(un)substituted heterocyclic, (un)substituted heteroaromatic,Ar—NHSO₂Ar, Ar—NHCOAr, H,

wherein X is selected from the group consisting of H, —SO₂-Camphor,—CO(CH₂)₆COOH, —CO(CH₂)₃CONHOH, —CH₂CH₂NHBoc, —CO(CH₂)₃COOH, —CH₂COOH,—CH₂CO-Leu-Phe-CH₂OH, —CH₂COOMe, 2-OH, 2-Me, 2-NHFmoc, 2-NHSO₂CH₂Ph,2-NHSO₂CH₃, 2-NHSO₂(4-tBu)Ph, 2-NHSO₂(4-NHAc)Ph, 2-NHSO₂(3-CF₃)Ph,2-NHSO₂(4-NO₂)Ph, 2-NHSO₂(4-OMe)Ph, 2-NHSO₂(4-Br)Ph, 2-NHSO₂(4-I)Ph,2-NHSO₂(4-Ph)Ph, 2-NHSO₂(4-OPh)Ph, 2-NHAc, 2-NHCO(4-tBu)Ph,3′-CH₂CH(CO₂Me)NH₂, 3′-CH₂CH(CO₂Me)NHFmoc, 3′-t-Bu, 3′-t-Bu, 3′-OH,3′-O(CH₂)₃CHCH₂, 3′-O(CH₂)₄Br, 3′-O(CH₂)₄NMe₂, 3′-O(CH₂)₄N₃,

3′-O(CH₂)₄NH₂, 3′-CF₃, 3′-CH₃, 3′-n-heptyl, 3′-n-nonyl, 3′-i-Bu,3′-i-Propyl, 3′-n-Propyl, 3′-n-Octyl, 3′-Et, 3′-n-pentyl, 3′-n-Bu,3′-n-hexyl, 3′-n-cyclopentyl, 3′-n-cyclohexyl, 3′-NHSO₂(4-tBu)Ph, 3′-Me,3-F, 3-CF₃, 4-NHSO₂(4-tBu)Ph, 4-OH and 4-t-Bu, and wherein Y is selectedfrom the group consisting of H, 4-OH 4-t-Bu, and 3′-NHSO₂(4-tBu)Ph; R₅is selected from the group consisting of O, NH and CH₂; and R₆ isselected from the group consisting of (un)substituted aryl,(un)substituted heterocyclic, (un)substituted heteroaromatic,Ar—NHSO₂Ar, Ar—NHCOAr, H, Ph, Me, —CH₂-(4-Cl)Ph, —CH₂CHCH₂₅-CH₂OH,CH₂COOH, CH₂OCH₂CHCH₂, —SO₂(4-O-n-Bu)Ph, —SO₂(4-O-Ph)Ph, —SO₂(4-NHAc)Ph,—SO₂(4-CH₂CH₂CO₂Me)Ph, —SO₂(4-OMe)Ph, —SO₂(4-t-Bu)Ph, and R₆—CN. Inanother aspect, R₄ and R₆ are covalently linked via

In accordance with a first example, diaryloxindole compounds arerepresented by Formula I, set forth below.

In at least certain examples, A is selected from the group consisting ofcarbocyclic aromatic ring, heterocyclic ring and heteroaromatic ring, R₁is selected from the group consisting of (halo)alky, (un)substituted(alkyl)aryl, halogen, CN, COOH, alkenyl, alkynyl, alkoxy andcycloalkoxy, R₂ is selected from the group consisting of (un)substitutedaryl, (un)substituted heterocyclic, (un)substituted heteroaromatic,Ar—NHSO₂Ar and Ar—NHCOAr, R₃ is selected from the group consisting of(un)substituted aryl, (un)substituted heterocyclic, (un)substitutedheteroaromatic, Ar—NHSO₂Ar and Ar—NHCOAr, R₄ is selected from the groupconsisting of (un)substituted aryl, (un)substituted heterocyclic,(un)substituted heteroaromatic, Ar—NHSO₂Ar and Ar—NHCOAr, X is selectedfrom the group consisting of unsubstituted or substituted nitrogen,oxygen, sulfur and carbon, Y is selected from the group consisting ofunsubstituted or substituted nitrogen, oxygen, sulfur and carbon; andn=0-4.

In accordance with a second example, diaryloxindole compounds arerepresented by Formula II, set forth below.

In at least certain examples, R₁ is selected from the group consistingof H, 5-bromo, 5-chloro, 7-bromo, 5-iodo, 5-OCF₃, 5-sulfonic acid,5-nitro, 4-bromo, 6-bromo, 5-ethyl, 7-ethyl, 4-ethyl, 6-ethyl, 7-iodo,5-fluoro, 7-COOH and 7-COOMe, and R₂ is H. In other examples, R₁ is 4-Cland R₂ is selected from the group consisting of7-formamide-N-[2-(4-Amino-phenyl)-ethyl], 7-COOH, 7-CH₂OH and7-CH₂—N-imidazole.

In accordance with a third example, diaryloxindole compounds arerepresented by

Formula III, set forth below.

In at least certain examples, X is selected from the group consisting ofoxygen, NH and CH₂, and R is selected from the group consisting of3′-CH₂CH(CO₂Me)NH₂, 3′-CH₂CH(CO₂Me)NHFmoc, 3′-t-Bu, 3′-t-Bu, 3′-OH,3′-O(CH₂)₃CHCH₂, 3′-O(CH₂)₄Br, 3′-O(CH₂)₄NMe₂, 3′-O(CH₂)₄N₃,

3′-O(CH₂)₄NH₂, 3′-CF₃, 3′-CH₃, 3′-n-heptyl, 3′-n-nonyl3′-i-Bu,3′-i-Propyl, 3′-n-Propyl, 3′-n-Octyl, 3′-Et, 3′-n-pentyl, 3′-n-Bu,3′-n-Hexyl, 3′-n-cyclopentyl, 3′-n-cyclohexyl, 4-NHSO₂(4-tBu)Ph and3′-NHSO₂(4-tBu)Ph.

In accordance with a fourth example, diaryloxindole compounds arerepresented by Formula IV, set forth below.

In at least certain examples, R is selected from the group consisting of4-F, 4-O(CH₂)₃CHCH₂, 3-Cl, 3-F, 3-Me, 2-Me and 2-OMe.

In accordance with a fifth example, diaryloxindole compounds arerepresented by Formula V, set forth below.

In at least certain examples, R is selected from the group consisting of—SO₂-camphor, —CO(CH₂)₆COOH, —CO(CH₂)₃CONHOH, —CH₂CH₂NHBoc,—CO(CH₂)₃COOH, —CH₂COOH, —CH₂CO-Leu-Phe-CH₂OH and —CH₂COOMe.

In accordance with a sixth example, diaryloxindole compounds arerepresented by Formula VI, set forth below.

In at least certain examples, R₁ is selected from the group consistingof 7-Br, 4-Cl, 5-Br, 5-NO₂, 5-I, 5-OMe, 5-Cl, 7-CF₃, 5-NH₂, 4-Br, 6-Br,H, 7-OMe, 7-I, 5-Et, 7-Et, 4-Et, 6-Et, 5-F, 5-N₃, 7-COOH, 4-Cl, 6-Cl and5-NHSO₂-1-napthyl, and R₂ is H. In other examples, R₁ is 4-Cl and R₂ is7-COOH.

In accordance with a seventh example, diaryloxindole compounds arerepresented by Formula VII, set forth below.

In at least certain examples, R₁ is selected from the group consistingof H, 2-OH, 3-O(CH₂)₄NMe₂, 3-O(CH₂)₄N₃, 3-t-Bu, 2-OMe, 3-OMe, 4-OH,4-n-propyl, 4-i-Bu, 4-n-Bu, 4-O(CH₂)₂OH, 4-OMe, 2-CH₂CO₂H and 4-t-Bu,and R₂ is H. In other examples, R₁ is selected from the group consistingof 2-OH, 4-OMe, 3-t-Bu, 2-OMe, 2-OH, 4-OH, 4-OPh, 4-Ph and 4-NMe₂, andR₂ is selected from the group consisting of 2-OH, 4-OH and 4-t-Bu.

In accordance with an eighth example, diaryloxindole compounds arerepresented by Formula VIII, set forth below.

In at least certain examples, R₁ is selected from the group consistingof H and 7-COOH, R₂ is selected from a group consisting of H, 2-Me,3′-Me, 3-F and 3-CF₃, and R₃ is selected from a group consisting of H,2-NHFmoc, 2-NHSO₂CH₂Ph, 2-NHSO₂CH₃, 2-NHSO₂(4-tBu)Ph, 2-NHSO₂(4-NHAc)Ph,2-NHSO₂(3-CF₃)Ph, 2-NHSO₂(4-NO₂)Ph, 2-NHSO₂(4-OMe)Ph, 2-NHSO₂(4-Br)Ph,2-NHSO₂(4-I)Ph, 2-NHSO₂(4-Ph)Ph, 2-NHSO₂(4-OPh)Ph, 2-NHAc,2-NHCO(4-tBu)Ph and 3′-NHSO₂(4-tBu)Ph.

In accordance with a ninth example, diaryloxindole compounds arerepresented by Formula IX, set forth below.

In at least certain examples, R is selected from the group consisting ofPh, Me, —CH₂-(4-Cl)Ph, —CH₂CHCH₂, —CH₂OH, CH₂COOH, CH₂OCH₂CHCH₂,—SO₂(4-O-n-Bu)Ph, —SO₂(4-O-Ph)Ph, —SO₂(4-NHAc)Ph, —SO₂(4-CH₂CH₂CO₂Me)Ph,—SO₂(4-OMe)Ph and —SO₂(4-t-Bu)Ph.

In accordance with a tenth example, diaryloxindole compounds arerepresented by Formulae X-XII, set forth below.

In accordance with an eleventh example, diaryloxindole compounds arerepresented by Formula XIII, set forth below.

In at least certain examples, R is selected from the group consisting of2-thiophene, —(CH₂)₁₁CH₃, —CH₂Ph, —CCH, -cyclohexyl and —C₆F₅(pentafluorophenyl).

In accordance with a twelfth example, diaryloxindole compounds arerepresented by Formula XIV, set forth below.

In at least certain examples, R is selected from the group consisting ofH, 3-Cl, 3-F, 3-Me and 3-t-Bu.

In accordance with other examples, diaryloxindole compounds include, butare not limited to, compounds comprising Formula I to Formula XIV, andthe active and/or partially active compounds set forth in FIGS. 4A-4Band Tables 1-14.

In at least certain examples, the compounds disclosed here can be usedin the treatment of cellular proliferative disorders, such as cancer.Treatment of cellular proliferative disorders is intended to include,but is not limited to, inhibition of proliferation including rapidproliferation. As used herein, the term “cellular proliferativedisorder” includes, but is not limited to, disorders characterized byundesirable or inappropriate proliferation of one or more subset(s) ofcells in a multicellular organism. The term “cancer” refers to varioustypes of malignant neoplasms, most of which can invade surroundingtissues, and may metastasize to different sites (see, for example, PDRMedical Dictionary 1st edition (1995), incorporated herein by referencein its entirety for all purposes). The terms “neoplasm” and “tumor”refer to an abnormal tissue that grows by cellular proliferation morerapidly than normal and continues to grow after the stimuli thatinitiated proliferation is removed. Id. Such abnormal tissue showspartial or complete lack of structural organization and functionalcoordination with the normal tissue which may be either benign (i.e.,benign tumor) or malignant (i.e., malignant tumor).

The language “treatment of cellular proliferative disorders” is intendedto include, but is not limited to, the prevention of the growth ofneoplasms in a subject or a reduction in the growth of pre-existingneoplasms in a subject. The inhibition also can be the inhibition of themetastasis of a neoplasm from one site to another. In certainembodiments, the neoplasms are sensitive to one or more diaryloxindolecompounds of the present invention. Examples of the types of neoplasmsintended to be encompassed by the present invention include, but are notlimited to, those neoplasms associated with cancers of the breast, skin,bone, prostate, ovaries, uterus, cervix, liver, lung, brain, larynx,gallbladder, pancreas, rectum, parathyroid, thyroid, adrenal gland,immune system, neural tissue, head and neck, colon, stomach, bronchi,and/or kidneys.

In accordance with certain other examples, methods for treating viralinfections are also disclosed. Treatment of viral infections is intendedto include, but is not limited to, the use of a diaryloxindole compounddescribed herein to prevent the initiation of viral protein synthesis.The term “viral infection,” as used herein, refers to one or more cellswhich have been infected with a virus, such as a DNA or RNA animalvirus. As used herein, RNA viruses include, but are not limited to,virus families such as picornaviridae (e.g., polioviruses), reoviridae(e.g., rotaviruses), togaviridae (e.g., encephalitis viruses, yellowfever virus, rubella virus), orthomyxoviridae (e.g., influenza viruses),Paramyxoviridae (e.g., respiratory syncytial virus, measles virus, mumpsvirus, parainfluenza virus), rhabdoviridae (e.g., rabies virus),coronaviridae, bunyaviridae, flaviviridae, filoviridae, arenaviridae,bunyaviridae, and retroviridae (e.g., human T-cell lymphotropic viruses(HTLV), human immunodeficiency viruses (HIV)). As used herein, DNAviruses include, but are not limited to, virus families such aspapovaviridae (e.g., papilloma viruses), adenoviridae (e.g.,adenovirus), herpesviridae (e.g., herpes simplex viruses), andpoxyiridae (e.g., variola viruses). In certain embodiments, the viralinfection is caused by hepatitis B virus, hepatitis C virus, and/or HIV.

In accordance with other examples, methods for treating disordersassociated with viral infections are disclosed. Treatment of one or moredisorders associated with viral infections is intended to include, butis not limited to, the use of a diaryloxindole compound described hereinto reduce or alleviate one or more symptoms of a viral infection. Asused herein, the term “disorders associated with viral infection” refersto the host's response to infection by one or more viruses. Suchresponses include, but are not limited to neurological symptoms (e.g.,encephalitis, meningoencephalitis, paralysis, myelopathy, neuropathy,aseptic meningitis, hemiparesis, dementia, dysphagia, lack of muscularcoordination, impaired vision, coma, and the like), wasting symptoms(e.g., inflammatory cell infiltration, perivascular cuffing of bloodvessels, demyelination, necrosis, reactive gliosis and the like),gastroenteritis symptoms (e.g., diarrhea, vomiting, cramps and thelike), hepatitis symptoms (nausea, vomiting, right upper quadrant pain,raised liver enzyme levels (e.g., AST, ALT and the like), jaundice andthe like), hemorrhagic fever symptoms (e.g., headache, fever, chillsbody pains, diarrhea, vomiting, dizziness, confusion, abnormal behavior,pharyngitis, conjunctivitis, red face, red neck, hemorrhage, organfailure and the like), oncogenic symptoms (e.g., sarcomas, leukemias andthe like, as well as “rare” malignancies, e.g., Kaposi's sarcoma, oralhairy leukoplasia, lymphomas and the like), immunodeficiency symptoms(e.g., opportunistic infections, wasting, rare malignancies,neurological disease, fever, diarrhea, skin rashes and the like),lesions (e.g., warts (e.g., common wart, flat wart, deep hyperkaratoticpalmoplantar wart, superficial mosaic type palmoplantar wart and thelike), epidermodysplasia, mucosal lesions, ulcers and the like), andsystemic symptoms (e.g., fever, chills, headache, muscle pain, bonepain, joint pain, pharyngitis, tonsillitis, sinusitis, otitis,bronchitis, pneumonia, bronchopneumonia, nausea, vomiting, increasedsalivation, rash, macules, lymphadenopothy, arthritis, ulcers,photosensitivity, weight loss, irritability, restlessness, anxiety,coma, death and the like). Disorders associated with viral infectionsare described in Fields Virology 4^(th) Ed. (2001) Lippincott, Williams& Wilkins, and the introduction to medical virology website(web.uct.ac.za/depts./mmi/jmoodie/introvi2.html), incorporated herein byreference in their entirety for all purposes.

In accordance with other examples, methods for treatingnon-proliferative, degenerative disorders associated with aberranttranslation initiation using a diaryloxindole compound described hereinto alleviate and/or reduce one or more symptoms associated with anon-proliferative, degenerative disorder are disclosed. Treatment ofnon-proliferative, degenerative diseases is intended to include, but isnot limited to, the use of diaryloxindole compounds. As used herein, theterm “non-proliferative degenerative disorder” is intended to include,but is not limited to, diseases characterized by a loss of function ofcells, tissues, and/or organs due to aberrant translation initiation.Non-proliferative degenerative disorders include, but are not limitedto, disorders such as Alzheimer's disease and insulin resistance.

The term “calcium releaser,” as used herein, refers to molecules whichcause a sustained depletion of intracellular Ca²⁺ stores and inhibittranslation initiation. Calcium releasers include, but are not limitedto, molecules such as clotrimazole (CLT), fatty acids such as EPA,diaryloxindole compounds of the present invention (including, but notlimited to compounds comprising Formula I to Formula XIV and the activeand/or partially active compounds set forth in FIGS. 4A-4B and Tables1-14), and the like.

In accordance with certain other examples, kits for treating one or more(1) proliferative disorders, (2) non-proliferative, degenerativedisorders, (3) viral infections, and/or (4) disorders associated withviral infections are provided. In one example, the kit may comprise oneor more diaryloxindole compounds, or a combination of one or morediaryloxindole compounds. In another example, the kit may comprise apharmaceutically acceptable carrier. In an additional example, the kitmay also include instructions for treating (1) proliferative disorders,(2) non-proliferative, degenerative disorders, (3) viral infections,and/or (4) disorders associated with viral infections. In some examples,the kit may also comprise, e.g., a buffering agent, a preservative, or aprotein stabilizing agent. In other examples, the kit may also contain acontrol sample or a series of control samples which can be assayed andcompared to the test sample contained. Other suitable components forincluding in the kit will be selected by the person of ordinary skill inthe art, given the benefit of this disclosure.

In accordance with certain examples, diaryloxindole compounds can beincorporated into pharmaceutical compositions suitable foradministration. Such compositions typically comprise the compoundsdisclosed here and a pharmaceutically acceptable carrier. As used hereinthe term “pharmaceutically acceptable carrier” is intended to includeany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

In accordance with certain examples, a pharmaceutical composition of theinvention is formulated to be compatible with its intended route ofadministration. Such pharmaceutical compositions may be administered byinhalation, transdermally, orally, rectally, transmucosally,intestinally, parenterally, intramuscularly, subcutaneously,intravenously or other suitable methods that will be readily selected bythe person of ordinary skill in the art, given the benefit of thisdisclosure. For example, solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerin, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampules,disposable syringes or multiple dose vials made of glass or plastic.

In accordance with other examples, pharmaceutical compositions suitablefor injectable use include sterile aqueous solutions (where watersoluble) or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersion. Forintravenous administration, suitable carriers include physiologicalsaline, bacteriostatic water, CREMPHOR EL™ (BASF, Parsippany, N.J.), orphosphate buffered saline (PBS). In all cases, the composition must besterile and should be fluid to the extent that easy syringabilityexists. It must be stable under the conditions of manufacture andstorage and must be preserved against the contaminating action ofmicroorganisms such as bacteria and fungi. The carrier can be a solventor dispersion medium containing, for example, water, ethanol, polyol(for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmanitol, sorbitol, sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent which delays absorption, forexample, aluminum monostearate and gelatin.

In accordance with other examples, sterile injectable solutions can beprepared by incorporating the active compound in the required amount inan appropriate solvent with one or a combination of ingredientsenumerated above, as required, followed by filtered sterilization.Generally, dispersions are prepared by incorporating the active compoundinto a sterile vehicle which contains a basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions,methods of preparation can be vacuum drying and freeze-drying whichyields a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof. Oralcompositions generally include an inert diluent or an edible carrier.They can be enclosed in gelatin capsules or compressed into tablets. Forthe purpose of oral therapeutic administration, the active compound canbe incorporated with excipients and used in the form of tablets,troches, or capsules. Oral compositions can also be prepared using afluid carrier for use as a mouthwash, wherein the compound in the fluidcarrier is applied orally and swished and expectorated or swallowed.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

In at least certain examples, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These may be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811, incorporated herein by reference in its entirety for allpurposes.

In accordance with certain examples, pharmaceutical compositions of theinvention comprise one or more diaryloxindole compounds covalentlylinked to a peptide (i.e., a polypeptide comprising two or more aminoacids) (FIGS. 4A-4B). Peptides may be assembled sequentially fromindividual amino acids or by linking suitable small peptide fragments.In sequential assembly, the peptide chain is extended stepwise, startingat the C-terminus, by one amino acid per step. In fragment coupling,fragments of different lengths can be linked together, and the fragmentscan also be obtained by sequential assembly from amino acids or byfragment coupling of still shorter peptides.

In both sequential assembly and fragment coupling it is necessary tolink the units (e.g., amino acids, peptides, compounds and the like) byforming an amide linkage, which can be accomplished via a variety ofenzymatic and chemical methods. The methods described herein forformation of peptidic amide linkages are also suitable for the formationof non-peptidic amide linkages.

Chemical methods for forming the amide linkage are described in detailin standard references on peptide chemistry, including Muller, Methodender organischen Chemie Vol. XV/2, 1-364, Thieme Verlag, Stuttgart,(1974); Stewart and Young, Solid Phase Peptide Synthesis, 31-34 and71-82, Pierce Chemical Company, Rockford, Ill. (1984); Bodanszky et al.,Peptide Synthesis, 85-128, John Wiley & Sons, New York, (1976); Practiceof Peptide Synthesis, M. Bodansky, A. Bodansky, Springer-Verlag, 1994and other standard works in peptide chemistry, incorporated herein byreference in their entirety for all purposes. Methods include the azidemethod, the symmetric and mixed anhydride method, the use of in situgenerated or preformed active esters, the use of urethane protectedN-carboxy anhydrides of amino acids and the formation of the amidelinkage using coupling reagents, such as dicyclohexylcarbodiimide (DCC),diisopropylcarbodiimide (DIC),1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), pivaloylchloride, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDCI), n-propane-phosphonic anhydride (PPA),N,N-bis(2-oxo-3-oxazolidinyl)amido phosphoryl chloride (BOP-Cl),bromo-tris-pyrrolidinophosphonium hexafluorophosphate (PyBrop),diphenylphosphoryl azide (DPPA), Castro's reagent (BOP, PyBop),O-benzotriazolyl-N,N,N′,N′-tetramethyluronium salts (HBTU),O-azabenzotriazolyl-N,N,N′,N′-tetramethyluronuim salts (TATU),diethylphosphoryl cyanide (DEPCN),2,5-diphenyl-2,3-dihydro-3-oxo-4-hydroxythiophene dioxide (Steglich'sreagent; HOTDO), 1,1′-carbonyldiimidazole (CDI) and the like. Thecoupling reagents can be employed alone or in combination with additivessuch as N,N-dimethyl-4-aminopyridine (DMAP), N-hydroxy-benzotriazole(HOBt), N-hydroxybenzotriazine (HOOBt), N-hydroxysuccinimide (HOSu),2-hydroxypyridine and the like.

In accordance with other examples, methods of modulating translationinitiation for therapeutic purposes are disclosed. In one example, amethod involves contacting a cell with an agent that inhibitstranslation initiation. An agent that inhibits translation initiationcan be any one of the compounds described herein, such as adiaryloxindole compound. In at least certain examples, the compoundmodulates the depletion of intracellular calcium stores. Methods ofmodulating translation initiation can be performed in vitro (e.g., byculturing a cell with the agent) or, alternatively, in vivo (e.g., byadministering the agent to a subject). Certain examples disclosed hereinare directed to methods of treating an individual afflicted with adisease or disorder characterized by aberrant translation initiation.Examples of such disorders are described herein. In one embodiment, themethod involves administering an agent (e.g., an agent identified by ascreening assay described herein), or combination of agents thatinhibits translation initiation. As used herein, an individual afflictedwith a disease or disorder is intended to include both human andnon-human mammals. Examples of non-human mammals include, but are notlimited to, non-human primates, horses, cows, goats, sheep, dogs, cats,mice, rats, hamsters, guinea pigs and the like.

The present invention provides for both prophylactic and therapeuticmethods of treating a subject for one or more (1) proliferativedisorders, (2) non-proliferative, degenerative disorders, (3) viralinfections, and/or (4) disorders associated with viral infection. In oneaspect, the invention provides a method for preventing in a subject, adisease or condition associated with one or more (1) proliferativedisorders, (2) non-proliferative, degenerative disorders, (3) viralinfections, and/or (4) disorders associated with viral infection, byadministering, to the subject one or more diaryloxindole compoundsdescribed herein to modulate one or more (1) proliferative disorders,(2) non-proliferative, degenerative disorders, (3) viral infections,and/or (4) disorders associated with viral infection. Administration ofa prophylactic agent can occur prior to the manifestation of symptoms,such that a disease or disorder is prevented or, alternatively, delayedin its progression.

Another aspect of the invention pertains to therapeutic methods oftreating one or more (1) proliferative disorders, (2) non-proliferative,degenerative disorders, (3) viral infections, and/or (4) disordersassociated with viral infection for therapeutic purposes. Accordingly,in an exemplary embodiment, a therapeutic method of the inventioninvolves contacting a subject with a diaryloxindole compound thattherapeutically treats one or more (1) proliferative disorders, (2)non-proliferative, degenerative disorders, (3) viral infections, and/or(4) disorders associated with viral infection.

One embodiment of the present invention involves a method of treating atranslation initiation-associated disease or disorder which includes thestep of administering a therapeutically and/or prophylacticallyeffective amount of an agent which inhibits translation initiation to asubject. In another embodiment, a subject is administered atherapeutically and/or prophylactically effective amount that iseffective to deplete intracellular calcium stores. As defined herein, atherapeutically and/or prophylactically effective amount of agent (i.e.,an effective dosage) ranges from about 0.001 to 30 mg/kg body weight,from about 0.01 to 25 mg/kg body weight, from about 0.1 to 20 mg/kg bodyweight, from about 1 to 10 mg/kg, from about 2 to 9 mg/kg, from about 3to 8 mg/kg, from about 4 to 7 mg/kg, or from about 5 to 6 mg/kg bodyweight. The skilled artisan will appreciate that certain factors mayinfluence the dosage required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Treatment of a subject with a therapeutically and/orprophylactically effective amount of an inhibitor can include a singletreatment or can include a series of treatments. It will also beappreciated that the effective dosage of in used for treatment mayincrease or decrease over the course of a particular treatment.

Example I General Synthetic Approach to Generate DiaryloxindoleCompounds

The general synthetic approaches to produce the diaryloxindole compoundsof the present invention are set forth in Tables 1-14. Tables 1-14depict biological data of compounds synthesized describing the generalsynthetic strategy for the scaffold set forth at the top of each table.The appropriate isatins were either commercially available orsynthesized starting from appropriate anilines.

A bioassay guided iterative approach was taken for the synthesis of thediaryloxindole compounds. The iterative approach involved an initialselection of compounds which were subjected to one or more bioassays,such as those described in Example III. Additional compounds were thensynthesized based on bioassay and/or structural (e.g., electronic andsteric nature of various diaryloxindole compound substituents) data. Thecompounds synthesized addressed the electronics and the sterics bysubstituting the three phenyl rings and the nitrogen with variousfunctional groups. The symmetric diphenyl compounds were obtained usingtriflic acid (TfOH) following previously reported procedures (Klumpp etal. (1998) J. Org. Chem., 63:4481, incorporated herein by reference inits entirety for all purposes), while the unsymmetrical compounds weresynthesized using a two step procedure. The first step involved aGrignard addition to isatin using appropriately substituted startingmaterials, and the key second step involved the generation of aquaternary center at the three position of the oxindole ring. This wasaccomplished by an acid catalyzed Friedel-Crafts type condensation ofstep B with the appropriate aromatic ring to generate diaryloxindoles(Hewawasam et al. (2002) Bioorganic & Medicinal Chemistry Letters,12:1023, incorporated herein by reference in its entirety for allpurposes). For activated ring systems that contained anelectron-donating group, p-toluene sulfonic acid in dichloroethaneallowed the condensation to occur smoothly, while the unactivated ringsystems required the use of triflic acid, which is much stronger thanp-toluene sulfonic acid.

As a non-limiting example, compounds 1272 and 1273 (set forth inTable 1) were synthesized starting from 3-bromoaniline, which uponcondensation with hydroxylamine and chloral hydrate followed byconcentrated sulfuric acid yielded an inseparable regioisomeric mixtureof 4- and 6-bromo isatins. The addition of the two phenyl groups to thethree position of the oxindole allowed easy chromatographic separationof the 4- and 6-bromo-3,3-diphenyloxindoles 1272 and 1273. Table 1 setsforth biological data for diaryloxindole compounds including, but notlimited to 1272 and 1273. For Tables 1-14, Ca²⁺ refers to Ca²⁺ releasefrom intracellular stores where a “+” denotes release and a “+/−”denotes inconclusive; eIF2α-P refers to phosphorylation of eIF2αmeasured by Western blot; SRB refers to the inhibitory concentration toreduce cell growth by 50% (IC₅₀) for growth inhibition of A549 lungcancer cells; and n.d. is not determined.

TABLE 1

Compound R₁ R₂ Ca²⁺ eIF2α-P SRB 1205 5-Br H + 2.05 9 1206 5-Cl H + 1.589 1207 7-Br H +/− 2.01 3 1263 5-OCF₃ H + n.d. 8 1264 5-I H + 2.53 9 12655-NO₂ H +/− 2.35 14 1266 5-SO₃H H − 1.62 9 1267 7-I H − 1.88 20 12687-Et H − 2.19 14 1269 5-Et H + 1.64 >20 1270 4-Et H + 1.68 >20 1271 6-EtH + 1.58 4 1272 4-Br H + 1.63 8 1273 6-Br H + 1.72 6 1350 5-F H + 1.16 91351 4-Cl 4-CO₂Me +/− 1.35 >20 1353 7-CO₂H H − 1.54 >20 1354 7-CO₂Me H +1.74 >20 1355 4-Cl

+/− 3.80 5 1392 4-Cl 7-CO₂H + 2.76 4 1398 4-Cl 7-CH₂OH − 1.48 14 14014-Cl 7-CH₂(N-Imidazole) +/− 2.56 >20

TABLE 2

Compound R X Ca²⁺ eIF2α-P SRB 1171 3′-CH₂CH(CO₂Me)NH₂ O + n.d  n.d. 11723′-CH₂CH(CO₂Me)NHFmoc O + 1.94 n.d. 1181 3′-t-Bu O + 5 3 1182 3′-5-BuH₂ + n.d. 12 1201 3′-OH O + n.d. 20 1202 3′-O(CH₂)₃CHCH₂ O + n.d. 6 12033′-O(CH₂)₄Br O + n.d. 8 1216 3′-O(CH₂)₄NMe₂ O +/− n.d. >20 12193′-O(CH₂)₄N₃ O + n.d. 16 1220

O +/− n.d. 16 1228 3′-O(CH₂)₄NH₂ O + n.d. 5 1243 3′-CF₃ O − n.d. >201278 3′-CH₃ O + 3.33 8 1279 3′-n-heptyl O +/− 3.49 3 1280 3′-n-nonyl O+/− 3.89 3 1281 3′-i-Bu O + 7.94 3 1282 3′-n-Propyl O + 4.96 4 12833′-n-Propyl O + 7.67 3 1284 3′-n-Octyl O − 2.91 3 1285 3′-Et O + 2.96 81286 3′-n-pentyl O + 8.60 3 1287 3′-n-Bu O + 10.08 3 1288 3′-n-Hexyl O +6.28 3 1289 3′-n-cyclopentyl O + 6.49 3 1290 3′-n-cyclohexyl O +/− 8.603 1624 4-NHSO₂(4-tBu)Ph O + n.d. <1 1638 3′-NHSO₂(4-tBu)Ph O + n.d. 3

TABLE 3

Compound R Ca²⁺ eIF2α-P SRB 1210 4-F + 1.86 4 1218 4-O(CH₂)₃CHCH₂ − 1.367 1221 3-Cl + 3.24 3 1223 3-F + 4.41 7 1224 3-Me + 1.74 9 1225 2-Me +1.69 4 1226 2-OMe + 2.22 3

TABLE 4

Compound R Ca²⁺ eIF2α-P SRB 1319 —SO₂-Camphor + n.d. 2 1321—CO(CH₂)₆COOH + n.d. 16 1323 —CO(CH₂)₃CONHOH + n.d. 3 1329 —CH₂CH₂NHBoc− 0.89 3 1396 —CO(CH₂)₃COOH − 1.72 7 1397 —CH₂COOH + 1.46 8 1400—CH₂CO-Leu-Phe-CH₂OH + 3.70 3 1402 —CH₂CO₂Me + 3.94 9

TABLE 5

Compound R₁ R₂ Ca²⁺ eIF2α-P SRB 1196 H 7-Br + n.d. 3 1209 H 4-Cl + 2.088 1211 H 5-Br + 1.14 3 1212 H 5-NO₂ + 1.28 4 1213 H 5-I +/− 2 3 1214 H5-OMe + 1.62 6 1215 H 5-Cl +/− 1.1 3 1230 H 7-CF₃ +/− 0.41 3 1231 H5-NH₂ + 1.46 14 1234 H 4-Br + 2.57 6 1235 H 6-Br + 3.2 4 12385-NHSO₂-1-napthyl H +/− n.d. 3 1240 H 7-OMe + 1.04 8 1253 H 7-I + 2.05 61254 H 5-Et + 2.12 4 1256 H 7-Et + 1.3 3 1257 H 4-Et + 1.11 10 1258 H6-Et + 3.13 4 1312 H 5-F + n.d. 4 1348 H 5-N₃ + n.d. 3 1408 H 7-COOH +2.08 >20 1418 4-Cl 7-COOH + 7.47 16 1428 H 4-Cl + n.d. 3 1429 H 6-Cl +n.d. 3

TABLE 6

Compound R₁ R₂ Ca²⁺ eIF2α-P SRB 1162 Me Me + n.d. 5 1163 2-OH; 4-OMe2-OH; 4-OMe + n.d. 10 1194 H H + 2.5 13 1195 2-OH; 3′-t-Bu 2-OH;3′-t-Bu + n.d. 3 1197 2-OH H + 1.50 14 1199 2-OH; 3-O(CH₂)₄NMe₂ H + n.d.3 1200 2-OH; 3-O(CH₂)₄N₃ H + n.d. 3 1208 4-Cl 2-OH + 1.42 10 1217 3-t-BuH + 1.5 4 1232 3-t-Bu 4-OH + n.d. 4 1237 2-OMe 4-t-Bu + 1.3 17 1239 2-OH4-t-Bu + 1.88 8 1240 4-OH 4-t-Bu + 1.04 14 1259 4-OH 4-OH − n.d. >201260 4-OPh 4-OH + n.d. 12 1261 4-Ph 4-OH + n.d. 10 1262 4-NMe₂ 4-OH +/−n.d. 10 1292 2-OMe H +/− 1.04 9 1293 3-OMe H +/− 1.30 14 1294 4-OMe H −1.52 10 1295 4-OH H + 1.66 16 1296 4-n-propyl H + n.d. 11 1297 4-i-BuH + 0.7 8 1298 4-n-Bu H +/− 0.45 8 1299 4-O(CH₂)₂OH H + 0.71 5 13274-OMe; 2-CH₂CO₂H H n.d. n.d. >20 1349 4-t-Bu H + 1.27 8

TABLE 7

Compound R₁ R₂ R₃ Ca²⁺ eIF2α-P SRB 1242 H 3-CF₃ H + 2.72 14 1244 H 3-FH + 1.38 12 1394 7-CO₂H 3′-Me 2-NHFmoc + 8.21 9 1395 H 3′-Me 2-NHFmoc −1.84 3 1405 H 3′-Me 2-NHSO₂CH₂Ph + 1.84 4 1417 7-CO₂H 3′-Me 2-NHSO₂CH₃ +0.96 >20 1427 H 3′-Me 2-NHSO₂CH₃ + n.d. 14 1430 H 3′-Me2-NHSO₂(4-tBu)Ph + 4.8 0.9 1431 7-CO₂H 3′-Me 2-NHSO₂(4-tBu)Ph − n.d. >201493 H 3′-Me 2-NHCO(4-tBu)Ph − 2.06 3 1512 H 3′-Me 2-NHSO₂(4-NHAc)Ph −1.01 4 1514 H 3′-Me 2-NHSO₂(3-CF₃)Ph + 1.65 3 1589 H 3′-Me2-NHSO₂(4-NO₂)Ph + 1 5 1590 H 3′-Me 2-NHSO₂(4-OMe)Ph + 1.12 5 1591 H3′-Me 2-NHSO₂(4-Br)Ph + 3.30 3 1592 H 3′-Me 2-NHSO₂(4-I)Ph + 5.19 3 1593H 3′-Me 2-NHSO₂(4-Ph)Ph − 2.22 4 1594 H 3′-Me 2-NHSO₂(4-OPh)Ph − 3.73 51623 H H 2-NHSO₂(4-tBu)Ph + n.d. <1 1639 H 3′-Me 2-NHAc n.d. n.d. >201648 H 2-Me 3′-NHSO₂(4-tBu)Ph + n.d. n.d.

TABLE 8

Compound R Ca²⁺ eIF2α-P SRB 1300 Ph − n.d. 8 1301 Me + n.d. 20 1302—CH₂-(4-Cl)Ph − n.d. 14 1303 —CH₂CHCH₂ +/− n.d. 15 1304 —CH₂OH − n.d. 101306 —SO₂(4-O-n-Bu)Ph − n.d. >20 1307 —SO₂(4-O-Ph)Ph − n.d. >20 1308—SO₂(4-NHAc)Ph − n.d. 11 1309 —SO₂(4-CH₂CH₂CO₂Me)Ph − n.d. 20 1310—SO₂(4-OMe)Ph − n.d. 20 1311 —SO₂(4-t-Bu)Ph − n.d. 11

TABLE 9

Compound R Ca²⁺ eIF2α-P SRB 1227 —CH₂OCH₂CHCH₂ + n.d. 3 1236 —CH₂CHCH₂+/− 2.23 3 1255 —Ph − n.d. 4 1339 —CH₂COOH − n.d. >20

TABLE 10

Compound Ca²⁺ eIF2α-P SRB 1291 − n.d. >20

TABLE 11

Compound Ca²⁺ eIF2α-P SRB 1320 − n.d. 4

TABLE 12

Compound Ca²⁺ eIF2α-P SRB 1313 + n.d. >20

TABLE 13

Compound R Ca²⁺ eIF2α-P SRB 1222 2-Thiophene + n.d. 9 1274 —(CH₂)₁₁CH₃ −0.91 3 1275 —CH₂Ph +/− 1.01 13 1276 —CCH + 0.75 13 1277 -cyclohexyl −1.08 10 1328 —C₆F₅ + n.d. 3

TABLE 14

Compound R Ca²⁺ eIF2α-P SRB 1314 3-Cl + n.d. >20 1315 H − n.d. >20 13163-F − n.d. >20 1317 3-Me − n.d. >20 1318 3-t-Bu + n.d. 2

Example II Methods of Synthesizing Intermediate and Final DiaryloxindoleCompounds

Compounds described herein were purified either by re-crystallization orby column chromatography, and were characterized by ¹H nuclear magneticresonance (NMR) and liquid-chromatography-atmospheric pressure chemicalionization-mass spectrometry (LC-APCI-MS).

As a non-limiting example, isatins and substituted isatins(intermediates set forth in Tables 1, 3, 5 and 7) were synthesized byheating a solution of appropriately protected aniline (0.1 moles),chloral hydrate (0.11 moles), hydroxylamine hydrochloride (0.2 moles) inconcentrated H₂SO₄ (12.5 g) and water (700 mL) to 95° C. for tenminutes. The solution was then kept at 4° C. overnight. The creamcolored isonitroso intermediate was filtered off, washed with water anddried. This compound was ground to a fine powder in a mortar and pestle,and added, with stirring, in portions over 30 minutes to concentratedH₂SO₄ (40 g) maintained at 60-65° C. The mixture was then heated at 95°C. for one hour and poured onto ice (300 g). The resulting solid wasfiltered to give the appropriate substituted isatin.

As another non-limiting example, 3-Aryl-3-hydroxy-substituted-oxindoles(intermediates set forth in tables 2-7 and 9-14) were synthesized byadding dropwise a 1M solution of alkyl or aryl magnesium halide (0.33mole) to an appropriately substituted isatin (0.1 mole) in 60 mL THF at0° C. The resulting mixture was allowed to warm to room temperature andlet spin at room temperature for twelve hours. The reaction mixture wasquenched with a saturated solution of NH₄Cl (100 mL) and diluted with100 mL of dichloromethane. The layers were separated, the organic layerwas washed with water followed by brine, dried over Na₂SO₄ and filtered.The filtrate was concentrated in vacuo to yield the product.

As another non-limiting example, symmetric substituted di-aryloxindoles(final compounds set forth in Tables 1 and 8) were synthesized by addingtwo mL of substituted benzene to a mixture of appropriately substitutedisatins (1 mmol) combined with three mL of freshly distilled triflicacid. The resulting mixture was stirred at room temperature for eighthours. The product mixture was poured over 25 g of ice and extractedinto either chloroform or toluene. The organic solution was washed withwater and then brine and dried over Na₂SO₄. Concentration in vacuoyielded the 3,3-diphenyl-substituted oxindoles.

As another non-limiting example, unsymmetrically substituteddi-aryloxindoles (final compounds set forth in Tables 2-7 and 9-13) weresynthesized by adding freshly distilled triflic acid (1 mmol) in 1 mLdichloromethane to a mixture of appropriately substituted3-aryl-3-hydroxy-oxindole (0.1 mmol) and appropriately substitutedbenzene dissolved in 1 mL dichloromethane. The reaction was maintainedat room temperature and monitored by thin-layer chromatography (TLC) andliquid chromatography-mass spectrometry (LCMS). Upon completion, themixture was poured over ice (25 g) and extracted with dichloromethane.The organic layer was dried and concentrated in vacuo to yield thedesired product.

As another non-limiting example, unsymmetrically substituteddi-aryloxindoles (final compounds set forth in Tables 2-7 and 9-13) werealso synthesized by adding p-toluenesulfonic acid (0.3 mmol) in 5 mLdichloromethane to a mixture of appropriately substituted3-Aryl-3-hydroxy-oxindole (0.1 mmol) and appropriately substitutedbenzene dissolved in 10 mL dichloroethane. The reaction was heated to95° C. and monitored by TLC and LCMS, upon completion the reactionmixture was cooled and filtered to remove the p-toluenesulfonic acid.The filtrate was concentrated in vacuo and purified to yield the desiredproduct.

As another non-limiting example,3-(5-tert-butyl-2-hydroxy-phenyl)-3-phenyl-1,3-dihydro-indol-2-one(compound 1181) was synthesized by adding dropwise a 1M solution ofphenylmagnesiumbromide (0.33 mole) to isatin (15 g, 0.1 mole) in 60 mLTHF at 0° C. The resulting mixture was allowed to warm to roomtemperature and let stir at room temperature for 12 hours. The reactionmixture was quenched with a saturated solution of NH₄Cl (100 mL) anddiluted with 100 mL of dichloromethane. The layers were separated, theorganic layer was washed with water followed by brine, dried overNa₂SO₄, and filtered. The filtrate was concentrated in vacuo to yield20.1 g (90%) of 3-hydroxy-3-phenyl-1,3-dihydro-indol-2-one. A mixture ofthe above solid (2.5 g, 10 mmole), p-t-Bu-phenol (1.5 g, 10 mmol),p-toluenesulfonic acid (3 g) in 40 mL dichloroethane, was heated to 95°C. for six hours. The mixture was cooled and filtered. The filtrate wasconcentrated in vacuo to yield 2.4 g (66%) of the desired product(compound 1181).

As another non-limiting example,4-tert-butyl-N-[5-hydroxy-4-methyl-2-(2-oxo-3-phenyl-2,3-dihydro-1H-indol-3-yl)-phenyl]-benzenesulfonamide(compound 1430) was synthesized by adding dropwise4-tert-butyl-benzenesulfonyl chloride (8 g, 34 mmol) in 5 mLdichloromethane to a stirring solution of 5-Amino-2-methyl-phenol (4 g,32 mmol) in 20 mL pyridine and letting the mixture spin overnight atroom temperature. The resulting mixture was concentrated in vacuo andthe product was purified by column chromatography to yield 8 g (78%) of4-tert-butyl-N-(3-hydroxy-4-methyl-phenyl)-benzenesulfonamide. A mixtureof 3-hydroxy-3-phenyl-1,3-dihydro-indol-2-one (3.9 g, 17 mmol),4-tert-butyl-N-(3-hydroxy-4-methyl-phenyl)-benzenesulfonamide (7 g, 22mmol) and p-toluenesulfonic acid (6.5 g, 32 mmol) in dichloroethane wasrefluxed for 12 hours. The resulting mixture was cooled to roomtemperature to yield a colorless solid. The solid was filtered andpurified using column chromatography to yield 8 g (88%) of the desiredproduct (compound 1430).

Example III Analysis of Diaryloxindole Compounds

Without intending to be bound by theory, the mechanism of action oftranslation initiation inhibitors is set forth in FIG. 1, and includesthe depletion (complete or partial depletion) of intracellular calcium(Ca²⁺) stores and phosphorylation of eIF2α. Compounds synthesized usingthe approaches described herein were screened for their ability topartially deplete intracellular Ca²⁺ stores using FURA-2AM loaded cells(Benzaquen et al. (1995) Nature Medicine, 1:534, incorporated herein byreference in its entirety for all purposes). A limited subset ofcompounds were further screened for their ability to deplete endoplasmicreticulum (ER) Ca²⁺. The Ca²⁺ content of the ER was monitored usingER-targeted, Ca²⁺ sensitive, recombinant proteins. These recombinantproteins emitted light by fluorescent resonance energy transfer (FRET)as a function of the Ca²⁺ content of the medium. Such assays arepreviously described (Miyawaki, A.; Mizuno, H.; Llopis, J.; Tsien, R.Y.; Jalink, K. Cameleons as cytosolic and intra-organellar calciumprobes, Oxford University Press: Oxford, London, 2001; pp 3-16,incorporated herein by reference in its entirety for all purposes).Compounds that depleted Ca²⁺ were further evaluated for their ability tophosphorylate eIF2α. eIF2α phosphorylation was measured by Western blotanalysis using the phospho-specific anti-eIF2α antibody, as previouslydescribed (Aktas et al. (1998) Proc. Natl. Acad. Sci. USA, 95:8280,incorporated herein by reference in its entirety for all purposes).Compounds that both depleted ER Ca²⁺ and phosphorylated eIF2α weretested in a lung cancer cell line (A549) for cell growth inhibition aspreviously described (Palakurthi et al. (2000) Cancer Research, 60:2919,incorporated herein by reference in its entirety for all purposes).

Compound 1181 (set forth in Table 2) was determined to be a Ca²⁺depleting translation initiation inhibitor, as it depleted ER-Ca²⁺,phosphorylated eIF2α, inhibited growth of a cancer cell line (A549), anddecreased squamous cell carcinoma tumor mass in mice (FIGS. 2A-2B, 3A-3Cand 5). Using compound 1181 for comparison, additional compounds werescreened for their ability to inhibit translation initiation in a Ca²⁺depletion-dependent manner (Tables 1 to 14).

One set of compounds (Table 1) had a variation of the functional groupsat the positions R₁ and R₂ on the oxindole phenyl ring. Substitutionsincluding, but not limited to, 5-Br, 4-Cl, 5-OCF₃ and 6-Et groups,resulted in compounds that depleted intracellular Ca²⁺. Compound 1392,which has 4-Cl and 7-CO₂H substitutions, showed improved eIF2αphosphorylation, which correlated with improved growth inhibition ofcancer cells. Compound 1398 (a negative control) exhibited reduced eIF2αphosphorylation relative to compound 1392, with a concomitant reducedpotency of tumor cell growth inhibition.

The positional effect of a bromo group was next investigated by placinga bromo group at 4, 5, 6 and 7 positions on the phenyl ring of theoxindole to generate compounds 1272, 1205, 1273 and 1207, respectively.The 7-substitution abrogated Ca²⁺ depletion activity, and none of thecompounds exhibited an increase in eIF2α phosphorylation relative tocompound 1392.

Compound 1430 (Table 7) released calcium and showed a 5-fold increase ineIF2α phosphorylation over DMSO at 5 μM. Ternary complex assay was8-fold over DMSO at 20 μM, and cell growth was inhibited at <1 μM. Theplasma levels for this in mice were estimated using liquidchromatography and mass spectrometry (LC-MS). Compound 1430 was orallyadministered at 160 mg/kg, 240 mg/kg, and 320 mg/kg to DBA/2J mice andthe plasma concentration was measured at three time points, one hour,three hours, and six hours, and is set forth in Table 15. The plasma wasisolated from the mice by heart-lung puncture and the samples wereprocessed and analyzed by LC-MS. The concentrations at each time anddose are in nmoles/mL of plasma (n.d.=not detected; X=not determined).

TABLE 15 Dose Time 320 mg/kg 240 mg/kg 160 mg/kg 1 hour 14.0 6.8 5.7 3hours 3.8 4.0 3.8 6 hours 2.8 n.d. X

Other embodiments will be evident to those of skill in the art. Itshould be understood that the foregoing description is provided forclarity only and is merely exemplary. The spirit and scope of thepresent invention are not limited to the above examples, but areencompassed by the following claims. All publications and patentapplications cited above are incorporated by reference herein in theirentirety for all purposes to the same extent as if each individualpublication or patent application were specifically indicated to be soincorporated by reference.

1. A method of inhibiting abnormal proliferation of cells comprisingcontacting the cells with a compound of formula III, formula IV, formulaVI, or formula VIII in a manner to phosphorylate eIF2α wherein thecompound of formula III is:

wherein X is selected from the group consisting of oxygen, NH and CH₂;and R is selected from the group consisting of 3′-CH₂CH(CO₂Me)NH₂,3′-CH₂CH(CO₂Me)NHFmoc, 3′-t-Bu, 3′-OH, 3′-O(CH₂)₃CHCH₂, 3′-O(CH₂)₄Br,3′-O(CH₂)₄NMe₂, 3′-O(CH₂)₄N₃,

3′-O(CH₂)₄NH₂, 3′-CF₃, 3′-CH₃, 3′-n-heptyl, 3′-n-nonyl, 3′-i-Bu,3′-i-propyl, 3′-n-propyl, 3′-n-octyl, 3′-Et, 3′-n-pentyl, 3′-n-Bu,3′-n-hexyl, 3′-n-cyclopentyl, 3′-n-cyclohexyl, 4-NHSO₂(4-tBu)Ph and3′-NHSO₂(4-tBu)Ph; the compound of formula IV is:

wherein R is selected from the group consisting of 4-F, 4-O(CH₂)₃CHCH₂,3-Cl, 3-F, 3-Me, 2-Me and 2-OMe; the compound of formula VI is:

wherein R₁ is selected from the group consisting of 7-Br, 4-Cl, 5-Br,5-NO₂, 5-I, 5-OMe, 5-Cl, 7-CF₃, 5-NH₂, 4-Br, 6-Br, H, 7-OMe, 7-I, 5-Et,7-Et, 4-Et, 6-Et, 5-F, 5-N₃, 7-COOH, 7-CO₂H, 4-Cl, 6-Cl and5-NHSO₂-1-napthyl; and R₂ is H or 7-CO₂H; and the compound of formulaVIII is:

wherein R₁ is selected from the group consisting of H and 7-CO₂H; R₂ isselected from the group consisting of H, 2-Me, 3′-Me, 3-F and 3-CF₃; andR₃ is selected from the group consisting of H, 2-NHFmoc, 2-NHSO₂CH₂Ph,2-NHSO₂CH₃, 2-NHSO₂(4-tBu)Ph, 2-NHSO₂(4-NHAc)Ph, 2-NHSO₂(3-CF₃)Ph,2-NHSO₂(4-NO₂)Ph, 2-NHSO₂(4-OMe)Ph, 2-NHSO₂(4-Br)Ph, 2-NHSO₂(4-I)Ph,2-NHSO₂(4-Ph)Ph, 2-NHSO₂(4-OPh)Ph, 2-NHAc, 2-NHCO(4-tBu)Ph and3′-NHSO₂(4-tBu)Ph.
 2. The method of claim 1, wherein the cells arecancer cells.
 3. The method of claim 1 carried out in an individual byadministration of the compound to the individual.
 4. The method of claim3 wherein the compound is administered by inhalation, transdermally,orally, rectally, transmucosally, intestinally, parenterally,intramuscularly, subcutaneously or intravenously.
 5. The method of claim1 wherein the compound is3-(5-tert-butyl-2-hydroxy-phenyl)-3-phenyl-1,3,-dihydro-indol-2-one or4-tert-butyl-N-(5-hydroxy-4-methyl-2-(2-oxo-3-phenylindolin-3-yl)phenyl)benzenesulfonamide.6. The method of claim 1 wherein the compound is3-(5-tert-butyl-2-hydroxy-phenyl)-3-phenyl-1,3,-dihydro-indol-2-one. 7.The method of claim 1 wherein the compound is 4-tert-butyl-N-(5-hydroxy-4-methyl-2-(2-oxo-3-phenylindolin-3-yl)phenyl)benzenesulfonamide.8. A method of inhibiting abnormal proliferation of cells comprisingcontacting the cells with 3-(5-tert-butyl-2-hydroxy-phenyl)-3-phenyl-1,3,-dihydro-indol-2-one or4-tert-butyl-N-(5-hydroxy-4-methyl-2-(2-oxo-3-phenylindolin-3-yl)phenyl)benzenesulfonamide in a manner to phosphorylate eIF2-α. 9.A method of inhibiting abnormal proliferation of cells in a human ornon-human mammal in need thereof, the method comprising administering tothe human or non-human mammal3-(5-tert-butyl-2-hydroxy-phenyl)-3-phenyl-1,3,-dihydro-indol-2-one or4-tert-butyl-N-(5-hydroxy-4-methyl-2-(2-oxo-3-phenylindolin-3-yl)phenyl)benzenesulfonamidein a manner to phosphorylate eIF2-α.
 10. A method of inhibiting abnormalproliferation of cells comprising contacting the cells with a compoundof formula III in a manner to phosphorylate eIF2α wherein the compoundof formula III is:

wherein X is selected from the group consisting of oxygen, NH and CH₂;and R is selected from the group consisting of 3′-CH₂CH(CO₂Me)NH₂,3′-CH₂CH(CO₂Me)NHFmoc, 3′-t-Bu, 3′-OH, 3′-O(CH₂)₃CHCH₂, 3-O(CH₂)₄Br,3′-O(CH₂)₄NMe₂, 3′-O(CH₂)₄N₃,

3′-O(CH₂)₄NH₂, 3′-CF₃, 3′-CH₃, 3′-n-heptyl, 3′-n-nonyl, 3′-i-Bu,3′-i-propyl, 3′-n-propyl, 3′-n-octyl, 3′-Et, 3′-n-pentyl, 3′-n-Bu,3′-n-hexyl, 3′-n-cyclopentyl, 3′-n-cyclohexyl, 4-NHSO₂(4-tBu)Ph and3′-NHSO₂(4-tBu)Ph.
 11. A method of inhibiting abnormal proliferation ofcells comprising contacting the cells with a compound of formula IV in amanner to phosphorylate eIF2α wherein the compound of formula IV is:

wherein R is selected from the group consisting of 4-F, 4-O(CH₂)₃CHCH₂,3-Cl, 3-F, 3-Me, 2-Me and 2-OMe.
 12. A method of inhibiting abnormalproliferation of cells comprising contacting the cells with a compoundof formula VI in a manner to phosphorylate eIF2α wherein the compound offormula VI is:

wherein R₁ is selected from the group consisting of 7-Br, 4-Cl, 5-Br,5-NO₂, 5-I, 5-OMe, 5-Cl, 7-CF₃, 5-NH₂, 4-Br, 6-Br, H, 7-OMe, 7-I, 5-Et,7-Et, 4-Et, 6-Et, 5-F, 5-N₃, 7-COOH, 7-CO₂H, 4-Cl, 6-Cl and5-NHSO₂-1-napthyl; and R₂ is H or 7-CO₂H.
 13. A method of inhibitingabnormal proliferation of cells comprising contacting the cells with acompound of formula VIII in a manner to phosphorylate eIF2α wherein thecompound of formula VIII is:

wherein R₁ is selected from the group consisting of H and 7-CO₂H; R₂ isselected from the group consisting of H, 2-Me, 3′-Me, 3-F and 3-CF₃; andR₃ is selected from the group consisting of H, 2-NHFmoc, 2-NHSO₂CH₂Ph,2-NHSO₂CH₃, 2-NHSO₂(4-tBu)Ph, 2-NHSO₂(4-NHAc)Ph, 2-NHSO₂(3-CF₃)Ph,2-NHSO₂(4-NO₂)Ph, 2-NHSO₂(4-OMe)Ph, 2-NHSO₂(4-Br)Ph, 2-NHSO₂(4-I)Ph,2-NHSO₂(4-Ph)Ph, 2-NHSO₂(4-OPh)Ph, 2-NHAc, 2-NHCO(4-tBu)Ph and3′-NHSO₂(4-tBu)Ph.